ABSTRACT: Gene expression analysis to compare control cells and sorted cells We transfected cDNA of human umbilical vein endothelial cells into rat myeloma cells, and sorted cells bound to patient IgG. Sorted cells were analyzed using the Affymetrix Human Genome U133 Plus 2.0 Array to investigate the cDNA inserted into the sorted cells. Array data was processed by Affymetrix GeneChip Command Console Software and Affymetrix Expression Console Software. No techinical replicates were performed.
Project description:Transcriptome analysis was done after warm renal ischemia-reperfusion injury (IRI) in a rat model. Earlier studies have shown a protective effect of prior unilateral nephrectomy (UNx) against IRI in the remaining, contralateral kidney compared to a non-neprectomized control group. We aimed at identifying the underlying molecular mechanisms. We used the Affymetrix Clariom D array (formerly known as RTA 1.0 st.) Array data was processed in the Affymetrix Console Software.
Project description:Total RNA was collected from DU145-control(scr) and DU145-miR-106a transfected cells. Gene expression analysis was performed by The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, Canada) using Affymetrix GeneChip Human Gene 2.0 ST array. Data were normalized using the default parameters in Affymetrix Expression Console Software 1.4. Genes downregulated 0.8-fold in miR-106a compared to control(scr) were identified as possible mRNA targets.
Project description:H226 cells transfected with DDX56 siRNA and control siRNA were incubated for 48 hours and total RNA isolated and used for the microarray analysis using Affymetrix Human U133 Plus 2.0 Arrays. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings.
Project description:Light-controlled in situ synthesis of DNA microarrays (Affymetrix GeneChip® Mouse Genome 430 2.0) were performed to determine the altered gene expression. Affymetrix algorithm and multiple analysis comparison software were used for assessing gene expression differences, and mRNAs that increased or decreased in the brains of NP(α-M)-treated mice relative to that in the brains of Blank-NP-treated mice were identified. For the assay, total RNA was extracted using Takara RNAiso Plus Kit (Cat#9109, Takara, DaLian, LiaoNing, CN) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). Array hybridization and wash was performed using GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instructions. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software12.6.1 (Agilent technologies, Santa Clara, CA, US).
Project description:mRNA samples from bone marrow CD34+ cells (HSC) from healthy donors after co-culture assays (Control condition, with hMSCs and CD34+ cells from healthy donors; and AML condition, with hMSCs-AML and CD34+ cells from donors) were amplified, labeled and hybridized to the ClariomTM S Array human (Thermo Scientific, USA). Normalization and analysis of microarray data was performed using the Transcriptome Analysis Console (TAC) software (Affymetrix, USA).
Project description:GFP expressing Plasmodium yoelii (Py-GFP) infected, or naïve cultured primary hepatocytes were FACS sorted and re-suspended in TRIzol and RNA purified with RNAeasy kit (Qiagen).RNA was assessed for purity and quality using an Agilent 2100 Bioanalyzer and transcriptome analysis performed on Affymetrix GeneChip Mouse Transcriptome array 1.0. Data output was analyzed and visualized using Affymetrix Expression Console 4.0.
Project description:GFP expressing Plasmodium yoelii (Py-GFP) infected, or naïve cultured primary hepatocytes were FACS sorted and re-suspended in TRIzol and RNA purified with RNAeasy kit (Qiagen).RNA was assessed for purity and quality using an Agilent 2100 Bioanalyzer and transcriptome analysis performed on Affymetrix GeneChip Mouse Transcriptome array 1.0. Data output was analyzed and visualized using Affymetrix Expression Console 4.0.
Project description:Natural Killer (NK) cells present natural cytotoxicity against tumor cells, although their activity is increased after activation.NK cell activation depends on a complex intracellular signaling process mediated by activating and inhibitory receptors and the functional outcome depends on the integration of the activating and inhibitory signals received. Soluble cytokines and/or ligands on target cells bind the NK cell receptors, and hence, influence the final NK cell response: attack versus ignorance. We used microarrays to detail the global programme of gene expression underlying NK cell activation by IL-2, a MHC-I-deficient target cell (K562)+IL-2 and an EBV-target cell (R69). PBLs from 4 different donors were activated by 100 U/ml IL-2; K562+IL-2 or R69 cells. After 5 days we obtained RNA and miRNA from naïve NK cells or from NK cells activated with the above mentioned stimuli, with more than 90% of purity. The 16 RNA samples were used to generate cDNA libraries that were hybridized on Human Gene 1.1ST arrays (Affymetrix) and analyzed with the Affymetrix Gene Chip Command Console Software v3.0 (AGCC 3.0, Affymetrix®) and the Expression Console Software v1.1 (Affymetrix®).
Project description:Natural Killer (NK) cells present natural cytotoxicity against tumor cells, although their activity is increased after activation. NK cell activation depends on a complex intracellular signaling process mediated by activating and inhibitory receptors and the functional outcome depends on the integration of the activating and inhibitory signals received. Soluble cytokines and/or ligands on target cells bind the NK cell receptors, and hence, influence the final NK cell response: attack versus ignorance. We used microarrays to detail the global programme of gene expression underlying NK cell activation by IL-2, a MHC-I-deficient target cell (K562)+IL-2 and an EBV-target cell (R69). PBLs from 4 different donors were activated by 100 U/ml IL-2; K562+IL-2 or R69 cells. After 5 days we obtained RNA and miRNA from naïve NK cells or from NK cells activated with the above mentioned stimuli, with more than 90% of purity. The 16 RNA samples were used to generate cDNA libraries that were hybridized on Human Gene 1.1ST arrays (Affymetrix) and analyzed with the Affymetrix Gene Chip Command Console Software v3.0 (AGCC 3.0, Affymetrix®) and the Expression Console Software v1.1 (Affymetrix®).
Project description:We probed the mechanism of cross-regulation of osmotic and heat stress responses by characterizing the effects of high osmolarity (0.3M vs. 0.0M NaCl) and temperature (43oC vs. 30oC) on the transcriptome of Escherichia coli K12 using E. coli Genome 2 Array (Affymetrix, Inc.). Independent array hybridizations were carried out for 3 biological replicates (independent cultures). Total RNA was extracted using a hot phenol-chloroform method. cDNA synthesis, fragmentation and labeling, and washing and scanning of E. coli GeneChip Arrays were performed according to the instructions of the manufacturer (Affymetrix Technical Manual, Affymetrix, Inc., USA). Labeled cDNA was hybridized to E. coli Genome 2 Array (Affymetrix, Inc.). Independent array hybridizations were carried out for 3 biological replicates (independent cultures) of each condition. A number of genes in the SoxRS and OxyR oxidative stress regulons were up-regulated by high osmolarity, high temperature, and/or by the combination of both stresses. This result could account for cross-protection of osmotic stress against oxidative stress. The trehalose biosynthetic genes were induced by both stresses, in accord with the proposed protective role of this disaccharide against thermal and oxidative damage. Keywords: Affymetrix microarrays